Determination of endometrial receptivity toward embryo implantation

ABSTRACT

Methods of diagnosing infertility in a mammal that include detecting the presence and level of a β 3  integrin subunit in an endometrium sample of a mammal at a plurality of stages of the endometrial cycle and correlating delayed appearance or a reduced level of β 3  integrin subunit expression with infertility are provided. Diagnostic kits useful in the practice of the methods of the invention are also provided.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/463,130, filed Jun. 16, 2003, which is a continuation of U.S.application Ser. No. 09/149,183, filed Sept. 8, 1998, now U.S. Pat. No.6,733,979, which is a continuation of U.S. application Ser. No.08/756,211, filed Nov. 25, 1996, now abandoned, which is a divisional ofU.S. application Ser. No. 08/400,270, filed Mar. 3, 1995, now U.S. Pat.No. 5,578,306, which is a continuation of U.S. application Ser. No.08/126,063, filed Nov. 1, 1993, now abandoned, which is a divisional ofU.S. application Ser. No. 07/897,706, filed Jun. 12, 1992, now U.S. Pat.No. 5,279,941. Benefit of priority under 35 U.S.C. §120 to each of theabove-noted applications is claimed. Each of these applications isincorporated by reference herein in its entirety as if full set forthbelow.

REFERENCE TO GOVERNMENT GRANTS

This work was supported in part by research grants from the BiomedicalResearch Support Grant Program, Division of Research Resources, theNational Institutes of Health, grant number B.R.S.B. S07-RR-05415-29.The United States Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Over the past decade, investigators have come to recognize theimportance of the extracellular matrix (ECM) in directing the growth,differentiation and function of the overlying epithelium. Getzenberg etal., “The Tissue Matrix: Cell Dynamics and Hormone Action”, EndocrineRev., 11:399-417 (1990). The interaction between cell and extracellularmatrix (or substratum) is mediated by several classes of cell adhesionmolecules, one of the most important being the integrins. Albelda etal., “Integrins and Other Cell Adhesion Molecules”, FESEB J.,4:2868-2880 (1990). Buck et al. “Integrin, a Transmembrane GlycoproteinComplex Mediating Cell-Substratum Adhesion”, J. Cell Sci. Suppl.,8:231-250 (1987). This diverse family of glycoprotein receptors isexpressed on the cell membrane as heterodimeric α and β subunits and isinvolved in both cell-cell and cell-substratum adhesion. Specificrecognition and binding of extracellular matrix (ECM) components such asfibronectin (FN), laminin (LM) and collagen (Col) transmit informationto the cytoskeletal structure, an interaction which may have major rolesin promoting hormone responsiveness and genomic activation. Burridge etal., “Focal Adhesions: Transmembrane Junctions Between the ExtracellularMatrix and the Cytoskeleton”, Ann. Rev. Cell. Biol. 4:487-525 (1988) andGetzenberg et al. supra.

Although extensive information exists about specific integrin proteins,for example, Hemler, M. E. “VLA Proteins in the Integrin Family:Structures, Functions and Their Role on Leukocytes”, Annu. Rev. Immunol:365-400 (1990), little is known concerning the distribution of thesereceptors in the female reproductive tract. In the uterus, theendometrium, composed of glandular epithelium and associated mesenchyme(stroma), maintains complex temporal and spatial functions in responseto the cyclic hormonal milieu. The search for morphological orbiochemical markers for uterine receptivity has been unsuccessful todate as reported by Rogers and Murphy, “Uterine Receptivity forImplantation: Human Studies”, in Blastocyst Implantation, Yoshinaga, K.ed., Serono Symposia, pp. 231-238 (1989). Once such markers areidentified, their role in endometrial phenomena including embryoimplantation, fertility, contraception and endometrial maturation andreceptivity can likely also be identified. Thus, as some integrinsappear to meet the criteria for markers of receptivity there is a greatneed for methods of detecting integrin cell adhesion molecules inendometrium.

SUMMARY OF THE INVENTION

The present invention is directed to methods of detecting receptivity ofendometrium to embryo implantation by detecting the β₃ subunit of theα_(v)/β₃ integrin in endometrium with a monoclonal antibody.

Methods of diagnosing fertility and methods of monitoring endometrialmaturation in a mammal are also provided by monitoring the appearance ofthe β₃ subunit of integrin in endometrium from a plurality of stages ofthe endometrial cycle. This is preferably done with a monoclonalantibody.

The present invention also provides methods of detecting the optimalwindow of embryo implantation in the endometrium by detecting the β₃subunit of integrin in an endometrial sample, preferably with amonoclonal antibody.

Further aspects of the invention include methods of preventing embryoimplantation by contacting the β₃ subunit of integrin in the endometriumwith neutralizing Fab antibody fragments to β₃. Methods of in vitrofertilization are also embodiments of the invention These comprisedetecting the β₃ subunit of integrin in an endometrial sample,fertilizing an egg in vitro, and introducing the zygote into the uterushaving endometrial tissue expressing the β₃ subunit.

Contraceptive and diagnostic kits are also contemplated hereby.

These and other aspects of the invention will become more apparent fromthe following detailed description when taken in conjunction with thefollowing figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F depict immunoperoxidase staining of normal endometrium.The photomicrographs depict the pattern of distribution for sixdifferent integrins that do not appear to change throughout themenstrual cycle. Dark areas represent positive staining, and light areasrepresent absence of stain (absence of specific integrin subunit).Immunohistochemical staining of the collagen/laminin receptor subunitsα₂(A), α₃(B), α₆(C), and β₄(D) shows prominent staining of epithelium(←) and microvessels (←) without significant stromal staining (*) forα₂, α₃, and β₄. Note basolateral staining α₈ and basal staining for β₄.Staining for fibronectin receptor subunits α₄(E), α₅(F) show predominantmesenchyme staining (*) with decreased epithelial staining (←). Theimmunoreactions (areas of dark staining) were developed byavidin-biotin-peroxidase complex using diaminobenzidine as a chromogen.For greater sensitivity, no counterstain was applied.

Magnification: 125×.

FIGS. 2A to 2C show photomicrographs of the immunohistochemical stainingfor the integrin subunit α₁ in proliferative vs. secretory endometrium.The staining in the glandular epithelium (←) was largely absent in theproliferative phase (A), and pronounced in all sections after menstrualcycle day 14 (B; day 20 endometrium). The microvasculature (←) stainingwas also pronounced, and did not change throughout the menstrual cycle.The staining noted in secretory endometrial glands was significantlyhigher than that of background (C). Magnification: 125×.

FIGS. 3A to 3D exhibit immunostaining of α_(v) and β₃ (the two pairingsubunits of the vitronectin receptor integrin) in proliferative phasevs. secretory phase endometrium. The staining intensity of α_(v) in theproliferative phase (A) was judged as “+” for the stromal cells (*) and“+” for glandular α_(v) (←). Immunostaining for α_(v) in day 22endometrium (B) demonstrates a significant increase in glandularstaining (example of “++” staining intensity). Likewise, the stainingfor β₃ was absent in proliferative epithelium (C; ←) and was notablyincreased in this day 22 secretory endometrium (D). Magnification: 125×.

FIGS. 4A and 4B show relative intensity of staining for the epithelialα_(v) and β₃ subunits in 35 endometrial samples throughout the menstrualcycle. The pattern of expression for α_(v) is shown in A, shows agradual increase in staining throughout the menstrual cycle. Incontrast, the pattern for β₃ in B, shows a more abrupt rise in thisintegrin subunit around day 20 of the menstrual cycle. Samples werestaged according to the last menstrual cycle. Sections were assigned ascore of 0 (−; negative), 1 (±; weak), 2 (+; moderate) or 3 (++;strong), by a blinded observer, and confirmed by a second observer.

FIGS. 5A to 5C depict staining intensity of epithelial β₃ in 12infertility patients with delayed endometrial maturation. Endometriumwas collected from women undergoing evaluation for infertility. Thebiopsies were separated into two groups based on the correlation betweenhistologic criteria and the menstrual cycle dating based on the time ofovulation and/or the subsequent menstrual period. Patients withendometrial biopsies 3 or more days “out of phase” (OOP group) werecompared with 25 endometrial biopsies that were “in phase” (Normal) andshown in A. Sections were assigned a score of 0 (−; negative), 1 (±;weak), 2 (+; moderate) or 3 (++; strong), based on the intensity ofepithelial β₃ staining. Examples of immunohistochemical staining of an“out of phase” biopsy (B) and a normal “in phase” sample (C) is includedto contrast the epithelial β₃ staining in each group. Magnification:400×.

FIGS. 6A to 6D exhibit immunoblot analysis of proliferative andsecretory endometrium, stained for the β₃ subunit. (A) Immunoblot ofplatelet extract (lane 1) compared with 2 samples from the early and midproliferative phase (lanes 2,3) and from the luteal phase (lane 4 and 5;days 23 and 26, respectively) demonstrates a band at approximately 95 kDmolecular weight, corresponding to β₃. Samples of endometrium werepartially digested with collagenase and the glandular elements obtained(B) using a modification of the methods of Satyaswaroop et al.,“Isolation and Culture of Human Endometrial Glands”, J. Clin. Endocr.Metab., 48:639-641 (1979). The glands appear as hollow structures freefrom surrounding stroma. Immunofluorescence of samples from lanes 3 and4 (C and D, respectively) corresponds to the absence or presence of the95 kD band in A. Magnification: 400×.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is directed to methods of detecting receptivity ofmammalian endometrium including obtaining a sample of endometrium,contacting the sample with a monoclonal antibody for the β₃ subunit ofintegrin and detecting the β₃ subunit.

For purposes of the present invention, the β₃ subunit may be β₃ alone orβ₃ in combination with another integrin subunit, α_(v), for example.

As used herein integrin is defined as a diverse class of glycoproteinreceptors expressed on the cell membrane. Integrins are cell adhesionmolecules of the immunoglobulin superfamily. Integrins are composed ofheterodimeric α and β subunits and are involved in cell-cell andcell-extracellular matrix adhesion. The integrin family is a broadlydistributed group of receptors composed of noncovalently associated α/βheterodimer pairs that mediate leukocyte-leukocyte andleukocyte-endothelial cell adhesion, as well as cellular interactionswith extracellular matrix components such as collagen, laminin,fibrinogen and fibronectin, and cell-cell interaction in organizedtissues.

While integrins are found on virtually all cell types (the exceptionbeing red blood cells), expression of integrin subunits varies from celltype to cell type. In human uterine endometrium, as determined herein,glandular epithelial cells express primarily α₂, α₃ and α₆ integrinsubunits, which are collagen laminin receptors. Stromal cells expresspredominantly α₅, a fibronectin receptor. The presence of α₁ onglandular epithelial cells is menstrual cycle specific, found onlyduring the secretory phase. Expression of both subunits of thevitronectin receptor, α_(v)/β₃, also undergoes menstrual cycle-specificchanges on endometrial epithelial cells. The expression of α_(v)increases throughout the menstrual cycle while the β₃ subunit appearsabruptly on menstrual cycle day 20 on luminal and glandular epithelialcells.

The present invention is directed to endometrium of the uterus of amammal. The uterine wall is largely smooth muscle or myometrium. Theendometrium, a glandular layer of variable thickness extremely sensitiveto the hormones estrogen/progesterone, lines the myometrium. Theendometrium is composed of several functional layers. The layer nearestthe myometrium is termed the basalis layer, and the layer closer to thesurface known as the functionalis. This tissue is made of epithelialcells, stromal (or mesenchymal) cells and endometrial leukocytes. Theepithelial cells are either glandular (forming glands beneath thesurface of the endometrium) or luminal (lining the surface of theendometrium). These different types of epithelium serve differentpurposes and staining patterns for different marker proteins are notalways the same between glandular and luminal. It is the luminal surfacethat would encounter the human embryo first and is thought to beinvolved in initial attachment. The endometrium of premenopausal girlsand postmenopausal women is atrophic due to the lack of the hormones,estrogen and progesterone. In the reproductive-aged woman, theendometrium undergoes cyclic developmental changes based on the ovariancycle of hormone release. The first day of menstruation is the first dayof the cycle; menstruation is generally completed by day 5. Theendometrial growth then resumes under the influence of estrogen andprogresses through the day 14, proliferative phase, and on to about day28. From day 14 to day 28 the endometrium also shows signs of decreasedgland growth and secretion, secretory phase, due largely to theinfluence of progesterone. During the follicular phase, while folliclesare growing in the ovary, and estrogen is the dominant hormone, theendometrium grows thicker. With ovulation (typically day 14 of a 28 daycycle) the women is exposed to estrogen plus progesterone. This iscalled the secretory or luteal phase, and is noted for a stereotypicseries of histologic changes that proceeds as the cycle continues. Thesehistologic changes are used by pathologists to date the endometrium, aprocess that remains controversial despite its use for the past 40years. There have been no reliable immunohistochemical markers reportedthat have proven utility in dating the endometrium.

Luteal phase dysfunction (LPD) is a term for developmental delay of theendometrium. It is a known cause of infertility, because of dysynchronybetween the fertilized egg and the endometrium. If an embryo is ready toattach but the endometrium is delayed, then pregnancy is not likely tooccur. The causes for LPD include inadequate hormonal output by theovary, and may implicate defective signaling from higher centers such asinadequate gonadotropic hormone output from the pituitary orhypothalamus. LPD is a known cause of infertility and spontaneousabortion and can be corrected with hormone augmentation.

Embryo implantation stages include: apposition—when the epithelial cellsof the embryo attach to the outer (luminal) epithelial cells of thematernal endometrial surface; adhesion; and invasion of trophoblast intothe underlying stroma where it establishes itself and begins to grow.Contact with maternal blood vessels is made to gain nutrients andoxygenated blood and to rid itself of waste products during the invasionstage. The stage of development that the embryo reaches at the time ofimplantation is the blastocyst stage, which occurs at the same time ashatching. There is evidence that hatching is required beforeimplantation occurs, perhaps because the embryo must have its epithelialcells exposed (out of the zona pellucida shell) to interact with thematernal cell layers. As set forth herein, this interaction occurs viaintegrins.

For purposes of the current invention, mammals include, but are notlimited to the Order Rodentia, such as mice; Order Logomorpha, such asrabbits; more particularly the Order Carnivora, including Felines (cats)and Canines (dogs); even more particularly the Order Artiodactyla,Bovines (cows) and Suines (pigs); and the Order Perissodactyla,including Equines (horses); and most particularly the Order Primates,Ceboids and Simoids (monkeys) and Anthropoids (humans and apes). Themammals of most preferred embodiments are humans.

Monoclonal antibodies useful in the practice of the invention includeany monoclonal antibodies having an affinity to or binding to the β₃subunit of integrin. An example of such a monoclonal antibody is SSA6.Monoclonal antibody SSA6 may be produced as described by Bennett et al.,PNAS, Vol. 80, 2417-2421 (1983).

Monoclonal antibodies which recognize β₃ combined with another integrinsubunit may also be used. One such monoclonal antibody is 23C6, whichmay be prepared according to the method of Davies et al., J. Cell Biol.,Vol. 109, 1817-1826 (1989). Immunostaining with monoclonal antibodiessuch as 23C6 (specific to the intact α_(v)/β₃ integrin, i.e. thevitronectin receptor) produces the identical pattern as SSA6 which onlymeasures the β₃ subunit. This demonstrates that while α_(v) specificantibodies measure all the α_(v) containing integrins, antibodies whichrecognize the intact α_(v)/β₃ integrin or the β₃ subunit can be used tostudy this integrin (the α_(v)/β₃ “vitronectin receptor”).

Other monoclonal antibodies can be used. The preparation of monoclonalantibodies is known to those in the art. Particularly, the method ofKohler and Milstein, Nature, 256: 495-497 (1975) may be used to producemonoclonal antibodies for use in the invention.

Methods of obtaining endometrial tissue samples for analysis include anysurgical and nonsurgical technique known in the art. Surgical methodsinclude, but are not limited to biopsy, dilation and curettage.Nonsurgical methods include, but are not limited to, uterine washingsand uterine brushings with immunocytochemical evaluation.

Methods of detecting β₃ in the endometrium include all methods ofidentifying glycoproteins known in the art. These methods include, butare not limited to, immunohistochemistry techniques such asimmunoblotting or Western blotting, immunoperoxidase staining,fluorescein labeling, diaminobenzadine and biotinylation.

Generally, immunohistochemistry involves staining cryosectioned tissuesamples. As used herein, endometrium samples may be cryosectioned toabout 4-8μ thick. Endometrium is contacted with primary antibody, suchas SSA6, followed by contact with secondary antibody, such asbiotinylated goat anti-mouse antibody. Endometrium is then incubated inavidin-conjugated horseradish peroxidase macromolecular complex followedby chromogen incubation, such as diaminobenzadine. Fluorescein may thenbe added to observe integrin distribution.

Immunoblotting involves the analysis of protein, here integrin, onsodium dodecylsulfate-polyacrylamide gel electrophoresis SDS-PAGE. Thegel is run under nonreducing conditions and the samples are transferredto a nitrocellulose membrane for example. The membrane is incubated inmedia containing primary antibody, such as SSA6. The filter is developedusing a secondary antibody, such as alkaline phosphatase-conjugated goatanti-mouse antibody.

The methods of diagnosing infertility and for detecting the window forembryo implantation in the endometrium of a mammal are also within thescope of the invention. As provided herein, the β₃ subunit of integrinappears at day 20 of the menstrual cycle. It is also provided hereinthat α_(v)/β₃ on endometrial epithelium binds fibronectin, vitronectinand osteopontin. These molecules may provide a bridge between theα_(v)/β₃ integrin of the endometrium and the embryo. Further, patientswith luteal phase dysfunction have delayed endometrial maturation,infertility and negative staining for β₃ on days 20 through 24. Thus,the optimal time for fertility may be determined by repetitively testingendometrial samples at a plurality of stages in the menstrual cycle. Assuch, screening for β₃ provides a method of diagnosing infertility andfor detecting the window of embryo implantation in the endometrium. Thewindow of implantation is that time when the endometrium of the uterusis available for embryo implantation. This window is preferably from day19 to day 23, and more preferably day 20 of the human menstrual cycle,marked by the expression of α_(v)/β₃ integrin.

Similar cycles are known for other mammals—it is within the ordinaryskill in the art to adopt the foregoing methodology to such cycles.

The present invention is also directed to methods of in vitrofertilization. Once the β₃ subunit of integrin is detected in an animalselected for pregnancy, a fertilizable egg (or eggs) from the same ordifferent animal could be replaced into the uterus to establishpregnancy. The egg and appropriate sperm are combined to produce azygote in vitro. For purposes of the invention, in vitro fertilizationmay take place in a petri dish, in a test tube or the like. In addition,in vitro fertilization may also refer to independently adding eggs andsperm to the fallopian tubes such that the zygote is formed therein. Inany event, the zygote is introduced to the uterus of the animal selectedfor pregnancy and monitored for implantation into the endometrium of theuterine wall.

Alternatively, the invention is directed to methods of preventing embryoimplantation. Such may be carried out by contacting the endometrium witha neutralizing Fab fragment specific for β₃. For purposes of the presentinvention, Fab fragments from monoclonal antibodies which bind β₃ arewithin the scope of the invention. Fab fragments include, but are notlimited to, Fab fragments from monoclonal antibodies SSA6 and 23C6. TheFab fragment may remain in vivo for a therapeutically effective time toprevent embryo implantation. The Fab fragment comprises the ligandbinding portion of a monoclonal antibody for β₃, i.e. the binding sitefor β₃. A neutralized Fab fragment is used in place of a typicalmonoclonal antibody to reduce the possibility of an inflammatoryreaction.

Contraception is a further embodiment of the invention. A contraceptivemay include a therapeutically effective amount of neutralizing Fabfragment monoclonal antibodies specific for β₃ in a pharmaceuticallyacceptable carrier, preferably adapted for intrauterine application.

Compounds which specifically block binding of the embryo to thisα_(v)/β₃ receptor are also included within the scope of the presentinvention. Examples include peptides containing the amino acid sequencearginine-glycine-aspartic acid, RGD (Pierschbacher et al., “SyntheticPeptide with Cell Attachment Activity of Fibronectin”, PNAS, Vol. 80,1224-1227 (1983)) which is the active binding site for the vitronectinreceptor. This sequence has been reported to block attachment ofpregnancy-derived cells (trophoblast) in vitro by researchers, Kao etal., “The Human Villous Cytotrophoblast: Interactions with ExtracellularMatrix Proteins, Endocrine Function, and Cytoplasmic Differentiation inthe Absence of Syncytium Formation”, Development, Vol. 130, 693-702(1988). Thus, a contraceptive containing the sequence RGD may beadministered locally to prevent embryo implantation.

Pharmaceutically acceptable carriers include, and are not limited to,vaginal suppositories, intrauterine devices (IUD), gels such as slowrelease formulation, for example, depo forms of hormones—microcrystalsinjected and slowly released into the systemic circulation or deliveredin silastic tubing. Contraceptive formulations would be administered inabout 10 μg/ml.

Methods of monitoring endometrial maturation is also within the scope ofthe present invention. The endometrium may be monitored for embryoreceptivity, embryo implantation, infertility, endometrial replenishmentand ovulation.

Diagnostic kits are also within the scope of this invention. Such kitsinclude monoclonal antibodies to rapidly detect β₃ in solution; anabsorbant detection device which contains pre-absorbed antibody againstβ₃ and to which uterine washings can be applied; a developer to make β₃visible when present.

The present invention is further described in the following examples.

These examples are not to be construed as limiting the scope of theappended claims.

EXAMPLES

Human Samples

Endometrium was obtained from 35 reproductive age women at the time ofhysterectomy. Tissue was obtained from the early proliferative (day 5)through late secretory phase (day 28) and all hysterectomies wereperformed for benign disease. Endometrial biopsies were performed onwomen as part of their evaluation for infertility. All patients werecycling normally and none had received hormones for at least 3 monthsprior to surgery. Dating of the endometrium was assessed according tothe criteria of Noyes et al., “Dating the Endometrium” Fertil. Steril.,1:3-8 (1950).

Endometrial biopsies were evaluated in the context of timing ofovulation and/or the onset of the next menstrual period. Samples werejudged as “out of phase” if histologic dating was delayed by 3 or moredays relative to the predicted day of the menstrual cycle. Proliferativeendometrium was categorized based on histology and on last menstrualperiod. Samples were transported on ice to the laboratory and were snapfrozen on dry ice and stored at −70° C.

Antibodies

Monoclonal antibodies (Mabs) PIH5, PIB5, PID6 specific to α₂, α₃, α₅subunits, respectively, were acquired from Drs. Elizabeth Wayner andWilliam Carter. Mabs TS2/7 and B-5H10 directed against the α₁ and α₄subunits, respectively were acquired from Dr. Martin Hemler. GoH3, aspecific Mab directed against α₆ was acquired from Dr. ArnoudSonnenberg. Mab SSA6 specific to the β₃ subunit was acquired from Drs.Joel Bennett and James Hoxie. Mab LM142 against α_(v) was acquired fromDr. David Cheresh. The β₄ antibody was acquired from Dr. Steven Kennel.The 23C6 antibody which recognizes β₃ attached to α_(v) was obtainedfrom Michael Horton.

Immunohistochemistry

Immunoperoxidase staining was performed on cryostat sections ofendometrium samples from throughout the menstrual cycle. Serialcryosections 4-8μ thick were placed onto poly-L lysine coated slides,fixed in −20° C. acetone for 10 minutes, and stained using VectastainElite® ABC kits (Vector Laboratories, Burlingame, Calif.).Diaminobenzidine (DAB; Sigma Chemical Co., St. Louis, Mo.) was used asthe chromogen. Primary antibody was placed on cryosections followingblocking with 1% bovine serum albumin in PBS, and allowed to bind atroom temperature for 1 hour. A phosphate buffered saline (PBS) pH 7.2 to7.4 rinse was followed by secondary antibody consisting of biotinylatedgoat anti-mouse antibody for 30 minutes. Following a PBS rinse, theendogenous peroxidases were quenched with a 30 minute incubation with0.3% H₂O₂ in absolute ethanol, followed by a 30 minute rehydration inPBS. Avidin:biotinylated horseradish peroxidase macromolecular complex(ABC) was then incubated on the sections for 30 minutes before addingdiaminobenzadine for 3 minutes to complete the reaction. Some sampleswere treated with 1:200 dilution of fluorescein-labeled anti-mouseantibodies for 1 hr., for immunofluorescent microscopy. Samples weresubsequently washed in PBS and mounted. The resulting staining wasevaluated on a Nikon microscope at low (100×) and higher (400×)magnification with or without fluorescence. Staining was judged asabsent (−), weak (±), moderate (+) or strong (++). Examples of each ispresented in FIG. 3. Photomicrographs were made using Kodak T-MAX 100ASA film.

Integrin Distribution in Normal Endothelium

The distribution of α₂, α₃, α₆, and β₄ subunits of integrins whichrecognize primarily collagen (Col) and/or laminin (LM) is shown in FIG.1 A-D. These subunits were present on glandular epithelium (←)throughout the menstrual cycle. Their distribution within theendometrium was typical of that seen for most epithelial tissues. The α₂and α₃ subunits were distributed around the entire circumference of thecells, while the α₆ and β₄ subunits appeared to be localized at thebasolateral surface, adjacent to the basement membrane (BM) of theendometrial glands. The expression of these subunits by the mesenchyme(*) was less pronounced. While moderate staining was seen for α₆ onstromal cells (FIG. 1C) very little staining was noted for β₄. Theexpression of the α₄ and α₅ subunits of integrins known to bindfibronectin (Table 1) was quite restricted. The α₄ subunit wasundetectable above background staining (compare FIG. 1E with FIG. 2C) ineither epithelium or mesenchyme. The α₅ subunit (FIG. 1F),representative of the classic fibronectin receptor was not seen on theepithelial components, but was strongly expressed in the mesenchyme,which is rich in fibronectin.

TABLE 1 DISTRIBUTION OF INTEGRINS BY LIGAND SPECIFICITY Ligands Integrinsubunit Collagen/Laminin Fibronectin/Vitronectin α₁/β₁ α₄/β₁ α₃/β₁α_(v)/β₁ α₂/β₁ α₅/β₁ α₆/β₁ α_(v)/β₃ α₆/β₁ α₆/β₄

The intensity of immunostaining for three other subunits of integrinswas found to change in a menstrual cycle-dependent manner.Immunostaining for α₁ in the proliferative phase (FIG. 2A) was onlyslightly above background levels (FIG. 2C). The intensity of stainingincreased throughout the secretory phase (FIG. 2B). This intensecircumferential staining was found on glandular and luminal epitheliumon all samples from day 15 to 28. Likewise, α_(v) was weakly expressedon both the epithelium and mesenchyme in the proliferative phase (FIG.3A) and staining increased gradually during the secretory phase to thelevel noted in FIG. 3B. During the proliferative phase β₃ staining wasonly present on the mesenchymal cells (FIG. 3C). Increased β₃ stainingwas apparent on the endometrial epithelium only after day 19 of themenstrual cycle (FIG. 3D) on the luminal as well as glandularepithelium, and was also present in a pericellular distribution. Incontrast, the basalis layer did not significantly stain for either α_(v)or β₃. This changing pattern of epithelial α_(v) and β₃ throughout thecycle was studied in 35 endometrial samples and is depicted graphicallyin FIGS. 4A and B.

Collagen/laminin receptors (col/LM) characterized by α₂, α₃ and α₆ wereuniformly expressed throughout the menstrual cycle, see Table 2.

The pericellular distribution of α₂ and α₃ subunits was distinctlydifferent from that of α₆ subunit. Characteristic of a laminin receptor,α₆ was concentrated on the basolateral surface. The β₄ subunit whichpairs with α₆, was also found distributed on the basolateral surface ofepithelial cells, and its distribution appeared even more restricted tothe basal pole. The α₅/β₁ integrin, a major fibronectin receptor, wasalso uniformly expressed throughout the menstrual cycle. Unlike thecollagen and laminin receptors, the distribution of α₅/β₁ was limited tothe mesenchyme.

The temporal pattern of distribution of α_(v) was varied. Immunostainingwas first detected prior to the secretory phase with an increase inintensity throughout the cycle. One subunit known to pair with α_(v) isβ₃. β₃ is not characteristically present on epithelial cells. The abruptappearance of the β₃ subunit after day 19 suggests that expression ofthe vitronectin receptor is regulated in human endometrium. Theincreased epithelial α_(v)/β₃ staining in normal cycles correlates to animplantation window within the secretory phase. While the physiologicbasis for the implantation window has not been previously established, aproposed role of integrins in the initial interaction between maternaland embryonic cells indicates an endometrial period of receptivity.

TABLE 2 DISTRIBUTION OF INTEGRIN SUBUNITS IN NORMAL ENDOMETRIUM DURINGTHE MENSTRUAL CYCLE Col/LM FN/VN Cell Type α₁ α₂ α₃ α₆ β₄ α₄ α₅ α_(v) β₃Epithelial ∘ • •  •^(b)  •^(b) ∘ ∘ ∘ ∘ proliferative early secretory • •• • • ∘ ∘ * ∘ late secretory • • • • • ∘ ∘ • • Stromal ∘ ∘ ∘ • ∘ ∘ • • •proliferative early secretory ∘ ∘ ∘ * ∘ ∘ * • • late secretory * ∘ ∘ • ∘∘ * • • b = basolateral distribution of staining • = + or + + staining *= ± staining ∘ = − stainingIntegrins in Discordant Endometrium.

The presence of the epithelial β₃ subunit appeared to be a consistentinternal marker of luteal phase maturation, and the timing of β₃expression correlated with the peri-implantation period or window ofembryo implantation. To investigate whether this phenomenon would beuseful in the clinical evaluation of endometrial biopsies,immunostaining was performed on luteal phase endometrial samples fromcycles which showed evidence of maturational delay. Endometrial biopsiesfrom 25 women who had concordance of menstrual and histologic dating(“Normal” group) were compared to 12 biopsies which were identified as≧3 days “out of phase” (OOP) based on either the time of ovulatory orthe subsequent menses. Samples were immunostained for α₁, α_(v) and β₃subunits. All biopsies were performed on days 20 to 24 of the menstrualcycle. In all instances, immunostaining for these three antigens waspresent on endometrial epithelia from the normal group. In biopsieswhich were delayed by 3 days or more, α₁ and α_(v) staining was present,but epithelial β₃ staining was absent. The comparison of β₃ stainingintensity in the two groups is shown in FIG. 5A. Accompanyingphotomicrographs of β₃ immunostaining from “out of phase” biopsies (OOP;B) and normal “in phase” biopsies (C) is included, which demonstratesthe discrepancy seen in β₃ staining. In subsequent treatment cycles, 2OOP patients underwent repeat biopsy during a normalized cycle at whichtime immunostaining for epithelial β₃ was present. This suggests thatthe lack of β₃ was not an intrinsic defect in the OOP group. Rather, thediscordant biopsies which lacked β₃ had not yet established themid-luteal phenotype of normal day 20 to 24 endometrium.

Cell Harvest and NP-40 Extraction

To further demonstrate that immunohistochemical staining accuratelyreflected changes in the expression of β₃ subunit on endometrialepithelium, immunoblots (Western blots) were performed on samples ofenriched endometrial glandular elements from proliferative and secretoryphase. Four samples of endometrium were obtained for the evaluation ofthe β₃ subunit in proliferative (n=2) and late secretory (n=2)endometrial epithelium. Each sample was placed in Dulbecco's modifiedEagle's medium (DMEM; Sigma, St. Louis, Mo.), supplemented with 10%fetal bovine serum (Flow Laboratories, McLean, Va.) glucose (4500 mg/L),Hepes buffer (25 mM), L-glutamine (584 mg/L), and sodium bicarbonate(3.7 gms/L). Endometrium was minced in a plastic petri dish prior toincubation with 6 mg of collagenase (type 1A, 550 units/mg; Sigma, StLouis, Mo.) for 2 hours at 37° C. utilizing modifications of theprocedures described by Satyaswaroop et al. in “Isolation and Culture ofHuman Endometrial Glands”, J. Clin. Endocr. Metab., 48:639-641 (1979).The resulting suspension was successively passed through a 250 μm sieveand a 38 μm seive (Newark Wire Cloth Co, Newark N.J.). The course (250μm) sieve removed undigested material, while the second retained theglandular elements and excluded the individual stromal and blood cells.After thorough rinsing, the glandular elements were obtained bybackwashing with 10 to 20 ml of DMEM. The isolated glandular structureswere then transferred to a 1.5 ml microfuge tube and centrifuged 3 times(82×g) for 2 minutes with intermittent washes with PBS. Membraneextracts were prepared by adding small volumes (100-200 μl) of 10 mMTris-acetate, pH 8.0, 0.5% NP-40, 0.5 mM Ca²⁺ (TNC) with 2 mM PMSF(phenyl methyl-sulfonyl fluoride) to the final pellet, pipetted andincubated on ice for 15 minutes. The lysate was centrifuged for 5minutes at 16,000×g in a microcentrifuge. The resulting supernatant wascalled NP-40 extract and was frozen at −70° C. until use. A portion ofthe original, undigested tissue was cryosectioned forimmunohistochemical localization of β₃.

Gel Electrophoresis and Immunoblots

The protein concentration of each NP-40 extract and an extract ofplatelets (positive control) was determined using technique described byLowry et al., “Protein Measurement with Folin Phenol Reagent”, J. Biol.Chem., 193:265-271 (1951). Samples with equal amounts of protein wereadded to electrophoresis sample buffer (62.5 mM Tris base, 2% SDS, 10%glycerol, pH 6.8). Samples were analyzed by SDS-PAGE using 6%poly-acrylamide gels, using non-reducing conditions described byLaemmli, U. K., “Cleavage of Structural Proteins During Assembly of theHead of Bacteriophage T4”, Nature, 227:680-685 (1970). The gel wastransferred to nitro-cellulose using a Biorad transfer apparatus(Biorad, Richmond, Calif.) and blocked with 4% BSA in PBS with 0.2% NaAzide for 1 hour. After addition of primary antibody (SSA6 supernatant)for 2 hours, the gels were developed using an alkalinephosphatase-conjugated secondary antibody (Promega Corp., Madison, Wis.)according to methods described by Albelda et al., “EndoCAM:A NovelEndothelial Cell-Cell Adhesion Molecule”, J. Cell Biol., 110:1227-1237(1990).

As shown in FIG. 6A, proliferative phase epithelial structures hadlittle to no immunostaining at 95 kD (lanes 2 and 3), compared to thepositive control (platelet extract; lane 1) or to samples from thesecretory phase (lanes 4 and 5) which showed strong staining for β₃. Theisolated endometrial glands appeared as tubular structures free ofsurrounding stroma (FIG. 6B). Immunofluorescent staining for β₃ fromsamples corresponding to lanes 3 and 4 (mid proliferative phase and day23, respectively) are shown in FIGS. 6C and D. Note the absence ofglandular staining in the proliferative sample, while both glandular andluminal immunostaining is obvious from the secretory phase. These dataconfirm that the expression of epithelial β₃ in human endometrium is acycle specific phenomenon.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

1. A method for diagnosing infertility in a mammal, comprising the stepsof: a) detecting the presence and level of a β₃ integrin subunit in anendometrium sample of a mammal at a plurality of stages of theendometrial cycle; and b) correlating delayed appearance or a reducedlevel of β₃ integrin subunit expression with infertility.
 2. The methodof claim 1, wherein expression is monitored during a plurality of stagesof the endometrial cycle.
 3. The method of claim 1, wherein the sampleof endometrium is obtained surgically.
 4. The method of claim 3, whereinthe sample is obtained by a biopsy or by a dilation and curettage. 5.The method of claim 1, wherein the sample of endometrium is obtainednonsurgically.
 6. The method of claim 5, wherein the sample is obtainedby a uterine washing or by a uterine brushing.
 7. The method of claim 1,wherein the β₃ subunit is combined with another integrin subunit.
 8. Themethod of claim 7, wherein the other integrin subunit is α_(v).
 9. Themethod of claim 1 wherein the stages of the endometrial cycle areselected from the group consisting of follicular phase and secretoryphase.
 10. The method of claim 1, wherein the endometrium sample isselected from the group consisting of: a) endometrium expressing a β₃integrin subunit; and b) endometrium not expressing a β₃ integrinsubunit.
 11. The method of claim 8, wherein the endometrium sample isselected from the group consisting of: a) endometrium expressing aα_(v)β₃ integrin; and b) endometrium not expressing a α_(v)β₃ integrin.12. The method of claim 1, wherein the endometrium sample is selectedfrom the group consisting of: a) endometrium tissue; b) endometriumepithelial cells; c) endometrium tissue extract; and d) endometriumepithelial cell extract.
 13. The method of claim 1, wherein theendometrium sample is partially digested with enzyme.
 14. The method ofclaim 13, wherein the enzyme is collagenase.
 15. The method of claim 1,wherein the β₃ integrin is detected by a technique selected from thegroup consisting of immunohistochemistry, immunoblotting, Westernblotting, Northern blotting, electrophoresis, immunoperoxidase staining,fluorescein labeling, biotinylation and reacting with diaminobenzadine.16. The method of claim 1, wherein the β₃ integrin is detected by itsmolecular weight.